PROJECT SUMMARY Hepatocellular carcinoma (HCC) accounts for nearly 29,000 deaths annually in the United States alone. However, the molecular mechanisms that drive HCC development remain elusive and current HCC therapies provide negligible clinical benefit. Factors that epigenetically silence an HCC tumor suppressor gene have the potential to promote tumorigenesis and thus may provide novel drug targets for HCC therapies. To discover such factors, we performed an innovative genome-wide human RNA interference (RNAi) screen to identify factors that mediate epigenetic silencing of the HCC tumor suppressor gene Hedgehog-Interacting Protein (HHIP). HHIP is a negative regulator of Sonic hedgehog (SHH) signaling and loss of HHIP due to epigenetic silencing aberrantly activates SHH signaling, which has been proposed to promote tumor growth in multiple cancers including HCC. One of the factors identified in our screen is Histone deacetylase 9 (HDAC9), a histone deacetylase involved in epigenetic gene silencing. Notably, epigenetic silencing of HHIP and HDAC9 overexpression occur frequently in HCC patient samples, supporting the clinical relevance of our results. Based on these results, we hypothesize that HDAC9 is a driver of HCC initiation and growth that functions by epigenetically silencing the tumor suppressor HHIP gene. The overall objective is to determine the role of HDAC9 in HCC initiation and growth, determine its mechanism-of-action and evaluate it as a drug target for HCC treatment. In Aim 1, we will determine the role of HDAC9 in driving HCC initiation and tumor growth. Towards this end, first using cell culture models of HCC, we will test if HDAC9 can transform immortalized hepatocytes. Additionally, we will test if HDAC9 can initiate HCC in vivo. For this purpose, we will use a genetically-defined mouse model of HCC initiation and a novel mouse model that we have developed to recapitulate human liver fibrosis. Liver fibrosis (also known as cirrhosis) is a cardinal feature of HCC and is shown to promote HCC initiation and tumor growth. In Aim 2, we will determine the mechanism-of-HDAC9- action. Our preliminary results allow us to predicts that tumor growth, which is inhibited following loss of HDAC9, can be restored by the simultaneous loss of HHIP or constitutive activation of SHH signaling. In this aim, experiments are proposed to rigorously test both of these predictions. We will also investigate other mechanisms by which HDAC9 may promote tumor growth, such as epigenetic silencing of a tumor suppressor gene other than HHIP. In Aim 3, we will evaluate pharmacological targeting of HDAC9 for HCC therapy. Towards this end, we will ascertain if in vivo pharmacological targeting using HDAC9 inhibitor TMP195 can effectively eradicate HCC tumor growth in vivo. For this purpose, we will use orthotopic mouse model of HCC tumor growth and progression. Collectively, the results of our experiments will elucidate a novel druggable epigenetic vulnerability pathway that drives HCC and validate the utility of HDAC9 targeting for effective HCC therapy.